Field of the Invention
This invention relates to protein crystallography, and in particular, it relates to single crystal quartz (or other crystal) chips used for protein crystal growth and X-ray diffraction data collection.
Description of the Related Art
Protein crystallography has greatly enriched our knowledgebase of protein structures by providing intricate details of many complicated molecular systems of life. Most recent advances are direct results of rapid progresses in structural genomics projects and wide applications of protein crystallography in many disciplines of life sciences. Protein Data Bank (PDB) is currently expanding at an annual rate greater than 10%. Many exciting new advances in biology demand even more powerful techniques to elucidate changes in protein structures as they function. For example, collection of solar energy by plants and photosynthetic bacteria; oxygenic photosynthesis to produce carbohydrates from carbon dioxide, water, and sun light; visual perception; and other environmental light perceptions to regulate biological processes and circadian rhythms all involve structural changes in proteins and their cofactors, many of which involve ultrafast molecular events. Better understanding of these fundamental processes at the molecular level would have broad and far-reaching impacts on areas of energy, agricultural, environmental, and biomedical sciences.
Indirect observations of protein structural changes have been inferred from comparisons of protein structures stabilized in different static states, for example, the open and closed states of ion channels. In an extreme case, nearly 300 hemoglobin structures have been compared simultaneously using an advanced numerical procedure. The resulting extensive reaction trajectory that twists and turns through several distinct states has shed light into the structural mechanism of cooperative oxygen binding and releasing.
More direct observations of structural changes require datasets collected before and after a deliberate alternation of the protein structure in crystal, such as soaking with a small molecular reagent. A more convenient, thus more practical, reagent to deliver into crystals is light. A temperature scanning technique based on cryocrystallography showed that multiple structural species of a red-light photoreceptor bacteriophytochrome could coexist under cryogenic temperatures. However, their populations shift towards the downstream of its reaction pathway as the temperature rises. A numerical deconvolution was needed to isolate the mixed heterogeneous structures.
A truly direct observation of protein structural changes during a reaction or a process demands experiments conducted in a “lights-camera-action!” style. Tremendous efforts at synchrotrons have advanced various techniques of Laue diffraction and time-resolved crystallography, also known as photocrystallography, to capture short-lived structural events that last not even as long as the duration of an electron bunch of a synchrotron, typically 100 ps (=10−10 s).
Most recently, ultrafast crystallography has become possible as high peak intensity, ultra-short pulses of hard X-rays as long as femtoseconds (fs=10−15 s) are produced by free electron lasers (XFEL) such as the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Lab. We are at a doorstep to direct observations of precursory events of electron and proton transfer, bond formation and rupture, isomerization, and their structural consequences in molecular systems as large as proteins. Mechanistic insights into important processes such as light harvesting, photosynthesis, biomass conversions, visual perception, and various environmental light perceptions rely on direct observations of fundamental chemical and biochemical reactions.
Despite significant progresses in recent years, a stubborn roadblock prevents much wider applications of time-resolved crystallography to many important systems even though light sensitive crystals are available. The chief difficulty arises from a direct conflict between the irreversible nature of many reactions or processes in crystalline states and the necessity of repetitive pump-probe cycles to accumulate signals and to complete a dataset. The technology developed over the past decades based on the classical time-resolved Laue diffraction at synchrotrons is unfortunately only applicable to rapidly reversible reactions, but most important biochemical reactions and biological processes are irreversible in crystalline states for two main reasons. 1) Lattice disorders due to large conformational changes associated with the reaction would significantly degrade the diffraction power of a crystal shortly after the first reaction trigger. 2) Radiation damage to crystals at room temperature also renders a reaction irreversible. Only those organic and protein systems capable of converting rapidly back to their dark states with minimal damage from laser and X-ray pulses have been thus far successfully studied by time-resolved crystallography. XFELs, which promise much greater peak intensity and far better time resolution, could only make this situation of irreversibility more severe by the diffract-and-destroy mode of data collection.
Although time-resolved holography, electron paramagnetic resonance spectroscopy, and coherent diffractive imaging have taken advantages of ultra-short XFEL pulses, numerous attempts at LCLS thus far have not yielded convincing changes in crystallographic electron density maps. Serial crystallography, using a liquid jet to deliver a train of nano to microcrystals into an XFEL beam, has demonstrated in several cases that good electron density maps could be derived from datasets merged from thousands of crystals. The ultra-short XFEL pulses have been exploited to race against radiation damage to nano and microcrystals, but not to capture ultrafast structural actions. While the liquid jet is able to deliver nano and microcrystals of hard-to-grow membrane proteins, it is not compatible with crystals of regular sizes. Nevertheless, the concept of serial crystallography provides an excellent guidance to future dynamic crystallography, except that a robotic delivering system for a large number of regular-sized crystals remains highly desirable.
In stark contrast to the repetitive pump-probe protocol, serial crystallography at room temperature is well suited for studies of irreversible reactions and processes. To accurately acquire difference structural signals, it is critically important to collect a time series of dark and light data points from a single crystal even if one series only covers a small fraction of a complete dataset. At a minimum, one pair of dark and light images is necessary from a same crystal. A large number of crystals would collectively contribute random slices of a complete dataset, each of which spans a full or partial period of a time series. A completely robotic system to deliver thousands of macroscopic crystals into an X-ray beam is a necessary component to achieve dynamic studies via serial crystallography including any ultrafast experiment at XFELs.